探讨结肠癌免疫微环境特征，筛选关键免疫相关基因并评估其预后价值，为免疫治疗靶点筛选及预后分层提供依据。

基于TCGA数据库514例结肠癌患者转录组及体细胞突变数据，利用CIBERSORT和ESTIMATE算法计算免疫评分、间质评分及ESTIMATE评分，随后，评估这些评分与患者生存结局（如总生存期）及各项临床病理参数之间的统计学关联。采用limma包筛选差异表达基因（DEGs），结合GO/KEGG富集分析明确功能；通过体细胞突变分析鉴定差异突变基因（DMGs），并与DEGs交叉验证，进一步分析关键基因与肿瘤浸润免疫细胞及预后的相关性，利用临床数据及细胞实验对结果进行验证。

高免疫评分与结肠癌患者总生存期显著正相关（低/高组：140，51，17，7，3 vs. 309，93，15，5，2；χ²=6.45，P=0.011），且免疫评分随肿瘤分期进展而显著下降。共筛选出627个免疫相关DEGs，富集于细胞因子受体相互作用等免疫通路。结合突变分析，识别出FN1和DOCK2为差异表达及突变的交叉关键基因：FN1高表达提示不良预后（χ²=6.44，P=0.011），其表达随分期增加而升高，且与淋巴结转移、远处转移呈显著正相关；DOCK2高表达具有保护作用（χ²=3.17，P=0.075），其表达随分期增加而下降，并与远处转移负相关。实验显示FN1在结肠癌组织和外周血中显著高表达（t=2.24，P=0.0257；t=3.54，P=0.0004），DOCK2显著低表达（t=−2.23，P=0.0260；t=−3.54，P<0.0001）。功能实验表明，FN1敲低可抑制癌细胞增殖（48 h：t=3.58，P=0.0006；60 h：t=3.66，P=0.0005）、侵袭（t=3.27，P=0.0404）及克隆形成（t=10.98，P=0.0001），而DOCK2敲低则增强上述能力（t=−1.94，P=0.0401；t=−9.14，P=0.0002；t=−3.95，P=0.0149）。

FN1和DOCK2通过调控肿瘤免疫微环境影响结肠癌进展及预后，FN1为风险因子，DOCK2为保护因子，可能成为免疫治疗潜在靶点及预后分层标志物。

To investigate the characteristics of the tumor immune microenvironment in colon cancer, identify key immune-related genes, and evaluate their prognostic value, thereby providing evidence for immunotherapy target screening and prognostic stratification.

Based on transcriptomic and somatic mutation data from 514 colon cancer patients in the TCGA database, CIBERSORT and ESTIMATE algorithms were used to calculate immune score, stromal score, and ESTIMATE score. The associations between these scores and patients’ clinical outcomes (e.g., overall survival) as well as various clinicopathological parameters were statistically evaluated. Differentially expressed genes (DEGs) were identified using the limma package and functionally annotated through GO/KEGG enrichment analyses. Somatic mutation analysis was used to identify differentially mutated genes (DMGs), which were cross-analyzed with DEGs to identify key genes associated with tumor-infiltrating immune cells and prognosis. Clinical data and cellular experiments were used to validate the results.

A high immune score was significantly positively correlated with overall survival in colon cancer patients (low/high immune score groups: 140, 51, 17, 7, 3 vs. 309, 93, 15, 5, 2; χ²=6.45, P=0.011), and the immune score significantly decreased with tumor stage progression. A total of 627 immune-related DEGs were identified, mainly enriched in immune pathways such as cytokine–cytokine receptor interaction. Combined with mutation analysis, FN1 and DOCK2 were identified as intersecting key genes with both differential expression and mutation: high expression of FN1 was associated with poor prognosis (P=0.011), increased with tumor stage, and was significantly positively correlated with lymph node and distant metastasis; high expression of DOCK2 had a protective effect (P=0.075), decreased with stage progression, and was negatively correlated with distant metastasis. Experiments showed that FN1 was significantly overexpressed in colon cancer tissues and peripheral blood (P=0.0257, 0.0004), while DOCK2 was significantly under expressed (P=0.0260, P<0.0001). Functional experiments demonstrated that knockdown of FN1 significantly inhibited colorectal cancer cell proliferation (48 h: t=3.58, P=0.0006; 60 h: t=3.66, P=0.0005), invasion (t=3.27, P=0.0404), and colony formation (t=10.98, P=0.0001). In contrast, DOCK2 knockdown promoted these malignant phenotypes (t=−1.94, P=0.0401; t=−9.14, P=0.0002; t=−3.95, P=0.0149).

FN1 and DOCK2 regulate colon cancer progression and prognosis by modulating the tumor immune microenvironment. FN1 serves as a risk factor, while DOCK2 acts as a protective factor, potentially serving as novel targets for immunotherapy and biomarkers for prognostic stratification.